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R A Harrison

Publications and source records attributed to R A Harrison.

At least 91 records · Page 5Linked to original sources
PubMed1994

Differential recognition by conglutinin and mannan-binding protein of N-glycans presented on neoglycolipids and glycoproteins with special reference to complement glycoprotein C3 and ribonuclease B.

Conglutinin and mannan-binding protein are serum proteins that have similar carbohydrate binding specificities toward high mannose-type oligosaccharides, and yet only conglutinin binds the complement glycoprotein iC3b, which contains oligosaccharides of this type. In the present study, the interactions of conglutinin and mannan-binding protein were evaluated with the complement glycoprotein C3, including various physiologically derived fragments of this glycoprotein, and neoglycolipids prepared from oligosaccharides released from C3 and its isolated alpha and beta chains. Several conclusions can be drawn. First, the interaction of conglutinin is profoundly influenced by the state of the protein moiety of the alpha chain in the vicinity of the glycosylation site Asn-917. Second, the binding to the C3-derived glycoprotein iC3b appears to be exclusively mediated through the Man8 or Man9 oligosaccharide on the alpha chain; there is no evidence for other N-linked oligosaccharides on C3 that are uniquely bound by conglutinin. Third, although conglutinin shows a more restricted binding relative to mannan-binding protein toward the oligosaccharides free of protein, it has a broader binding pattern toward the oligosaccharides as presented on C3-derived glycoproteins. From these and additional observations with RNase B, which contains high mannose-type oligosaccharides at Asn-34, it is clear that the protein moieties of these glycoproteins markedly influence the presentation of the oligosaccharides such that biological specificity is mediated by the commonly occurring high mannose-type oligosaccharides in the context of specific carrier proteins.

Blood Proteins↗
PubMed1994

Structure of a soluble, glycosylated form of the human complement regulatory protein CD59.

BACKGROUND: CD59 is a cell-surface glycoprotein that protects host cells from complement-mediated lysis by binding to and preventing the normal functioning of the complement proteins C8 and/or C9 which form part of a membrane penetrating assembly called the membrane attack complex. CD59 has no structural similarity to other complement proteins, but is an example of a plasma protein domain type found also in murine Ly-6 proteins and the urokinase-type plasminogen activator receptor. RESULTS: CD59 was purified from human urine, retaining the N-glycan and at least some of the non-lipid component of the glycosylphosphatidylinositol membrane anchor. The three-dimensional structure of the protein component has been determined in the presence of the carbohydrate groups using two-dimensional NMR spectroscopy. The protein structure is well defined by the NMR data (root mean square deviation from the mean structure of 0.65 A for backbone atoms and no distance constraint violations greater than 0.4 A). Structure calculations were also carried out to model the orientation of the N-acetylglucosamine residue that is directly linked to Asn18. CONCLUSIONS: The main features of the protein structure are two antiparallel beta-sheets (a central one with three strands and another with two), a short helix that packs against the three-stranded beta-sheet, and a carboxy-terminal region that, although lacking regular secondary structure, is well defined and packs against the three-stranded beta-sheet, on the opposite face to the helix. We have used the structure, in combination with existing biochemical data, to identify residues that may be involved in C8 binding.

Amino Acid Sequence↗
PubMed1994

Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma.

During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Importance of bicarbonate/CO2 for fertilization of pig oocytes in vitro, and synergism with caffeine.

Fertilization of pig oocytes was performed in vitro in modified Tyrode's media in which either HEPES or bicarbonate/CO2, or both, were included as buffer systems; caffeine (2 mM) was also included in some of the media because it is a reported stimulant of fertilization. The composition of the bicarbonate-containing media was designed so as to maintain the same pH and osmolality as bicarbonate-free media. The inclusion of bicarbonate during gamete co-incubation in caffeine-containing medium led to high levels of fertilization (66% of 238 mature oocytes were fertilized). However, essentially no fertilization occurred if bicarbonate was replaced with HEPES (0.7% of 146 oocytes were fertilized; significantly different, P < 0.001). Inclusion of HEPES in bicarbonate-containing medium during gamete co-incubation did not affect fertilization, showing that HEPES did not exert an inhibitory effect. Omission of bicarbonate during sperm preincubation also did not affect fertilization. If caffeine was included in bicarbonate-containing medium, 73% of 311 oocytes were fertilized whereas if caffeine was omitted only 14% of 326 oocytes were fertilized (significantly different, P < 0.001). In the absence of bicarbonate, when fertilization was very low, caffeine had no stimulatory effect. The results indicate that bicarbonate is essential for pig fertilization in vitro, but that caffeine exerts a synergistic stimulatory effect.

Animals↗
PubMed1993

A hinge region mutation in C1-inhibitor (Ala436-->Thr) results in nonsubstrate-like behavior and in polymerization of the molecule.

C1-inhibitor(Mo), a dysfunctional C1-inhibitor molecule produced in two kindred with type II hereditary angioedema, has a mutation at the P10 position (Ala436 to Thr). Like most serpins with hinge region mutations (P14, P12, P10), C1-inhibitor(Mo) loses its inhibitory activity. However, unlike the other hinge region mutations, this mutant is not converted to a substrate. As shown by nondenaturing gel electrophoresis, gel filtration, sucrose density gradient ultracentrifugation, and electron microscopy, C1-inhibitor(Mo) exists in both monomeric and multimeric forms. Polymerization probably results from reactive center loop insertion into the A sheet of an adjacent molecule. Native C1-inhibitor(Mo) was shown to have a thermal stability profile intermediate to those of intact and of cleaved normal C1-inhibitor. Native C1-inhibitor(Mo) did not bind to monoclonal antibody KII, which binds only to reactive center-cleaved normal C1-inhibitor. It did, however, react with monoclonal antibody KOK12, which recognizes complexed or cleaved C1-inhibitor but not intact normal C1-inhibitor. Native C1-inhibitor(Mo), therefore, exists in a conformation similar to the complexed form of normal C1-inhibitor.

Alanine↗
PubMed1993

Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations.

Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged ("dead") cells. If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.

Animals↗
PubMed1993

Chymotrypsin inhibitory activity of normal C1-inhibitor and a P1 Arg to His mutant: evidence for the presence of overlapping reactive centers.

C1-inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1-inhibitor, normal rabbit C1-inhibitor, and P1 Arg to His mutant human C1-inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate-resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1-inhibitor (3.4 x 10(4) compared with 7.3 x 10(3)). The carboxy-terminal peptide of normal human C1-inhibitor, derived from the dissociated protease-inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with alpha 2-antiplasmin, C1-inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg-specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike alpha 2-antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1-inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.

Amino Acid Sequence↗
PubMed1993

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.

Antigens, CD↗
PubMed1993

C1 inhibitor.

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Amino Acid Sequence↗
PubMed1993

Rapid isolation and biochemical characterization of rat C1 and C1q.

Using a human IgG-Sepharose column to which rabbit anti-human IgG was bound (rabbit anti-human/human IgG-Sepharose), human and rat C1 or C1q were isolated from serum in a single step, and the C1q further purified to homogeneity by FPLC. This procedure allowed the rapid isolation of haemolytically active C1 or C1q, with a yield equal to or greater than published methods. The availability of human and rat C1q allowed comparison of the two molecules, revealing differences in their mobility on SDS-PAGE as well as on agarose gel electrophoresis. Amino terminal sequence analysis demonstrated greater than 78% residue identity between rat C1q A, B and C chains and the published human and mouse sequences. Similar amino acid compositions suggest that the homology extends throughout the molecules. In addition to the major A:B and C:C dimer bands, rat, unlike human C1q, contained minor dimer species. These may reflect heterogeneity in glycosylation and or lysine and proline hydroxylation.

Amino Acid Sequence↗
PubMed1993

Contamination of surgeons' glove fingertips during surgical operations.

The external surfaces of surgeons' gloves were examined for contamination by bacteria during the course of surgical operations. Of 31 operations studied the surgeons' glove surfaces yielded bacteria at the end of the operation in 16 cases. Bacterial contamination increased during the course of the operation. The surgeons' fingertip skin and nail beds were cultured after surgical hand scrub and drying, but before donning gloves; bacteria were recovered from these sites in 10 of 21 and 13 of 21 cases, respectively.

Colony Count, Microbial↗
PubMed1993

The sheep analogue of human CD59: purification and characterization of its complement inhibitory activity.

An inhibitor of the membrane attack complex of complement was isolated from the membranes of sheep erythrocytes. Fast protein liquid chromatography (FPLC) and affinity purification procedures for this sheep complement-inhibiting protein (SCIP) both yielded a pure protein with an apparent M(r) of 19,000 under reducing and non-reducing conditions. Incubation of the denatured protein with neuraminidase and Endo-F reduced the apparent M(r) to 18,000 and 15,000 respectively, while treatment with O-deglycosidase or phosphatidylinositol-specific phospholipase C (PIPLC) did not affect the apparent M(r). SCIP was detectable on erythrocytes and lymphocytes but not on platelets and could partially be removed by PIPLC treatment. Deglycosylation of the pure protein markedly reduced and PIPLC treatment abolished its activity. A monoclonal antibody (mAb) raised against sheep complement-inhibiting protein (SCIP) enhanced the susceptibility of sheep erythrocytes to lysis by homologous complement. SCIP inhibited complement after the stage of C5b-7 formation. Amino-terminal protein sequence was obtained and was shown to be similar to that of human CD59. All these features suggest that SCIP is the sheep equivalent of human CD59. Human CD59 has been reported to be species selective in that it inhibits complement from relatively few species. However, SCIP efficiently inhibited lysis of guinea-pig erythrocytes by complement from a wide range of species tested indicating that it is a potent and non-selective inhibitor of the membrane attack complex of complement (MAC).

Amino Acid Sequence↗
PubMed1992

Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement C1 Inactivator Proteins↗
PubMed1992

Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement.

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

Amino Acid Sequence↗
PubMed1992

A dysfunctional C1 inhibitor protein with a new reactive center mutation (Arg-444-->Leu).

A P1 mutation (Arg-444-->Leu) was identified in a dysfunctional C1 inhibitor from a patient with type 2 hereditary angioneurotic edema. The mutation was defined at the level of the protein (by sequence analysis of the Pseudomonas aeruginosa elastase-derived reactive center peptide), and the mRNA (CGC-->CTC) (by sequence analysis of PCR-amplified DNA).

Amino Acid Sequence↗
PubMed1992

The role of diacylglycerol in the exocytosis of the sperm acrosome. Studies using diacylglycerol lipase and diacylglycerol kinase inhibitors and exogenous diacylglycerols.

When ram spermatozoa were treated with Ca2+ and the ionophore A23187 to induce acrosomal exocytosis, a rise in diacylglycerol (DAG) mass was observed, concomitant with a rapid breakdown of [32P]P1-labelled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and a rise in [32P]Pi-labelled phosphatidate. Inclusion of the DAG lipase inhibitor RHC 80267 resulted in further but biphasic increases in DAG; there was an increasing accumulation of DAG with concentrations of RHC 80267 up to 10 microM, whereas higher concentrations produced lessening accumulation. Inclusion of RHC 80267 in the ionophore induction system also resulted in significant accelerations of the onset of exocytosis. In spermatozoa stimulated with Ca2+/A23187 and the DAG kinase inhibitor R59022, a similar increase in DAG levels together with stimulation of acrosomal exocytosis were observed. Preincubation of spermatozoa with sn-1-oleoyl-2-acetylglycerol, rac-1-oleoyl-2-acetylglycerol, sn-1,2-dioctanoylglycerol and sn-1,3-dioctanoylglycerol before treatment with Ca2+/A23187 resulted in a dose-dependent stimulation of exocytosis by all these isomers. Neomycin inhibited Ca2+/A23187-induced generation of DAG together with polyphosphoinositide breakdown, as well as acrosomal exocytosis. Inclusion of exogenous DAG, however, overcame the inhibitory effect of neomycin on exocytosis. Our results suggest that DAG has a key role in acrosomal exocytosis and that it acts as a messenger rather than as a substrate from which other active metabolites are generated. The lack of stereospecificity shown by the exogenous DAGs implies that DAG does not act by stimulating protein kinase C, but the metabolite's actual target in the sperm cell is as yet unclear.

Acrosome↗
PubMed1992

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.

Complement C9↗